Background: In the last 15 years, over 3 million patients in the USA have been diagnosed with prostate cancer;approximately 1 million have undergone radical prostatectomy. The success of this surgery is measured by complete removal of the cancer-harboring gland and preservation of nerves that control sexual function. These nerves are minute and intermingle with fat cells and vascular tissue on the prostate capsule. Occasionally, the space around the nerves is used as an escape route by cancerous cells. Thus the inability to differentiate cancerous cells from nerves can result in the incomplete removal of the cancer and/or postoperative impotence due to the damage or excision of the nerves. The goal of this proposal is to provide surgeons access to real time tissue recognition tools that can better differentiate normal cells from cancerous ones and accurately identify and save the adjacent nerves, all while the surgery is in progress. Hypothesis: Multiphoton Microscopy (MPM) methods can be used to accurately visualize and differentiate prostate cancer from adjacent nerve fibers in real time. Specific Aims: Specific aim 1: We will test the hypothesis that MPM/SHG enables real-time imaging with sufficient resolution for tissue discrimination in the rat prostate. A training period will be accompanied by ex-vivo imaging of excised tissue from freshly euthanized rats (aim 1.1), followed by in vivo imaging of live, anesthetized rats (aim 1.2);all tissue identification results will be compared to those obtained by histological analysis. Specific aim 2: We will test the hypothesis that MPM/SHG imaging can be used for intravital imaging of the prostate and associated tissue in a rat prostate cancer model. Results will again be quantified and compared to those obtained by histopathological analysis of the same tissue. Specific aim 3: We will test the hypothesis that live imaging using MPM/SHG will not cause damage to the cavernous nerve or associated tissue by performing a safety assessment following intravital MPM/SHG imaging in rats. Continued function of nerves imaged by MPM/SHG will be quantitatively compared to unimaged nerves using a standard measure of erectile activity. Study Design: This application brings together experts in robotic prostatectomy (AT), MPM, SHG, and advanced optical microscopy(SM, FRM, WWW). We will standardize our imaging conditions and test our ability to identify all relevant tissue types (prostatic capsule, cavernous nerve, prostatic acinar cells, fat, arteries, and veins)by MPM/SHG imaging in a rat model. We propose to achieve this aim in two steps: Ex vivo imaging of tissue excised from euthanized rats and in vivo imaging in anesthetized rats. The identity of all tissues will be confirmed by standard histological analysis of the same specimen, and the statistical reliability of MPM identification will be assessed.We will extend these studies to a rat prostate cancer model. We will also test thehypothesis that MPM/SHG imaging in a live animal does not cause irreversible damage to the prostatic tissue or nerves by performing cavernous nerve stimulation six weeks after imaging. No exogenous dyes will be used, which will make the future translation of this technology to intra-operative use in humans via MP endoscopy much more straightforward. Endpoints: By the end of this funding period, we expect to have sufficient preliminary data to be ready to translate these techniques to human subjects via introperative use of a multiphoton endoscope currently being developed by our collaborators. Our findings will serve as a foundation for testing this MPM endoscope in actual clinical settings Impact: We anticipate that these studies, involving both animals and human surgical specimens, will lead to the development of methodologies applicable during surgery. We envision that live imaging during surgery will greatly reduce negative outcomes of radical prostatectomies.This technology may also be extended to other nerve-sparing pelvic oncologic surgeries, eg. radical cystectomy, extirpative colorectal and gynaecologic pelvic procedures, and retroperitoneal lymph node dissection. The dynamic, high-resolution optical images achieved could empower cancer surgeons to better distinguish tissue morphology during surgery, leading to improved functional outcomes and quality of life for their patients.